Method for stabilizing nitrilase activity and preserving microbial cells

ABSTRACT

A method for preserving immobilized or unimmobilized microbial cells having nitrilase activity and for stabilizing the nitrilase activity of unimmobilized or immobilized microbial cells has been developed. Aqueous suspensions containing at least 100 mM bicarbonate, carbonate, or carbamate salts limit microbial contamination of the stored enzyme catalyst, as well as stabilize the desired nitrilase activity of the unimmobilized or immobilized cells. Microorganisms which are characterized by an nitrilase activity and are stabilized and preserved by this method include  Acidovorax facilis  72-PF-15 (ATCC 55747),  Acidovorax facilis  72-PF-17 (ATCC 55745),  Acidovorax facilis  72W (ATCC 55746), and transformed microbial cells having nitrilase activity, the host cells transformed with  Acidovorax facilis  72W nitrilase activity. Especially preferred is an embodiment using ammonium carbamate as the inorganic salt.

FIELD OF THE INVENTION

[0001] The present invention relates to the field of microbiology andmolecular biology. More specifically, a method has been developed tostabilize the nitrilase activity of unimmobilized or immobilizedmicrobial cells and to preserve the integrity of the microbial cells,where the unimmobilized or immobilized microbial cells are stored in anaqueous solution containing from about 0.100 M to the saturationconcentration of an inorganic salt of bicarbonate, carbonate, andcarbamate, including ammonium, sodium, and potassium salts ofbicarbonate, carbonate, and carbamate.

BACKGROUND OF THE INVENTION

[0002] The occurrence of nitrile hydrolyzing enzymes has been widelydescribed. Within this family of enzymes, two broad classes aregenerally recognized. The first includes the nitrile hydratases, whichcatalyze the addition of one molecule of water to the nitrile, resultingin formation of the corresponding amide:

[0003] The second group includes the nitrilases which catalyze addingtwo molecules of water to the nitrile resulting in the direct formationof the corresponding carboxylic acid plus ammonia, without theintermediate formation of the corresponding amide:

[0004] Cells containing aromatic or aliphatic nitrilases have been usedto directly convert a nitrile to the corresponding carboxylic acidammonium salt in aqueous solution without the intermediate formation ofan amide (Kobayashi et al., FEMS Microbiol Lett. 120:217-234 (1994)).Cells having nitrilase activity include Rhodococcus rhodochrous K22(Kobayashi et al., Tetrahedron 46:5587-5590 (1990); Kobayashi et al., J.Bacteriology 172:4807-4815 (1990)), Acidovorax facilis 72W (Gavagan etal., Appl. Microbiol. Biotechnol. 52:654-659 (1999)), Comamonastestosteroni (U.S. Pat. No. 5,629,190 and U.S. Pat. No. 5,635,391;Lévy-Schil et al., Gene 161:15-20 (1995)), Rhodococcus rhodochrous NCIMB11216 (Gradley et al., Biotechnology Lett. 16:41-46 (1994);Bengis-Garber et al., Appl. Microbiol. Biotechnol. 32:11-16 (1989)), andRhodococcus rhodochrous J1 (Kobayashi et al., Eur. J. Biochem.182:349-356 (1989)). Of those nitrilases that have been sequenced, aunique conserved active-site cysteine has been identified as thefunctional group responsible for enzymatic nitrile hydration (Lévy-Schilet al., Gene 161:15-20 (1995); Cowan et al., Extremophiles 2:207-216(1998)). The typical method for stabilizing the cysteine-catalyzednitrilase activity has been to include mM concentrations ofdithiothreitol (DTT) and/or ethylenediamine-tetraacetic acid (EDTA) inthe aqueous buffer containing the isolated enzyme or whole cell.

[0005] A method for preserving suspensions of cells or immobilized cellshas been described in EP 0707061 A1, where the storage solution containsat least one inorganic salt selected from the group consisting ofphosphates, borates, sulfates, sulfites, and hydrochlorides. Types ofsalts included sodium, potassium, and ammonium salts. The concentrationof these salts ranged from 100 mM to the saturation concentration of theinorganic salt. The use of these inorganic salts to preserve thenitrilase activity of Gordona terrae MA-1 was demonstrated. Japanesepatent application JP 10042885 A2 describes the production of amidesand/or organic acids by treating nitrites with culture media, cells,cell preparations, enzymes, or enzyme preparations from microorganismshaving nitrile-hydrolyzing activities in aqueous reaction mixturecontaining H₂CO₃ or carbonate salts, where the inclusion of H₂CO₃ orcarbonate salts is reported to increase the observed activity of thenitrile-hydrolyzing enzyme towards the nitrile; no disclosure of animprovement in the stability of the nitrile-hydrolyzing activity of thecells over time when stored was made.

[0006] Methods for the preservation of nitrile hydration activity ofcells containing a nitrile hydratase (where an aliphatic nitrile isenzymatically converted to an amide without subsequent conversion of theamide to the corresponding carboxylic acid ammonium salt) are describedin U.S. Pat. No. 4,931,391 and U.S. Pat. No. 4,900,672. These methodsfor the stable preservation of nitrile hydratase activity ofunimmobilized or immobilized microorganisms add at least one compoundselected from the group consisting of nitrites, amides, or organic acidsto a suspension of the unimmobilized or immobilized cells.

[0007] U.S. Pat. No. 4,343,900 describes a process to produce acrylamidefrom acrylonitrile using a microorganism having a nitrilasic activity(that is, a nitrile hydratase) which includes in the reaction mixture analkali metal carbonate or bicarbonate. The alkali metal carbonate orbicarbonate is added to prevent a loss of enzyme activity that occursdue to a swelling of fixed cells during the reaction, and this loss ofactivity is not normally observed when physiological saline or phosphatebuffer is added to the reaction mixture. In this same method, theaddition of alkali metal carbonate or bicarbonate to reaction mixturesfor the production of acrylamide from acrylonitrile is preferably donein combination with the addition of an organic carboxylic acid, therebymaintaining the enzymatic activity for a long period of time whileacrylonitrile is converted to acrylamide.

[0008] The problems to be solved are to stabilize nitrilase activity andto reduce microbial contamination or putrefaction in suspensions ofunimmobilized or immobilized cells. Different methods have been used tostabilize cells having either nitrile hydratase or nitrilase activity,where these two enzyme activities are produced by different types ofenzymes which have different mechanisms for catalyzing nitrilehydrolysis to amides or acids, respectively. An examination of theeffect of various carboxylic acid salts on the storage stability ofnitrilase activity did not prevent the loss of nitrilase activity withtime as was seen with nitrile hydratase. Similarly, the use of variousinorganic salts previously disclosed as preserving the nitrilase ornitrile hydratase of microorganisms also did not stabilize the nitrilaseactivity of the microbial cells of the present invention upon storage asaqueous suspensions.

SUMMARY OF THE INVENTION

[0009] The invention solves the stated problem. It is a method forpreserving microbial cells having nitrilase activity and for stabilizingthe nitrilase activity thereof, the method comprising adding to anaqueous suspension of immobilized or unimmobilized microbial cellshaving nitrilase activity at least one compound selected from the groupconsisting of inorganic carbonate salts, inorganic bicarbonate salts,and inorganic carbamate salts, wherein the resulting total concentrationof the inorganic salts in the aqueous suspension ranges from 100 mM tothe saturation concentration of the inorganic salts. The microbial cellshaving nitrilase activity are selected from the group consisting ofAcidovorax facilis 72W (ATCC 55746), Acidovorax facilis 72-PF-15 (ATCC55747) and Acidovorax facilis 72-PF-17 (ATCC 55745). Additionally,transformed microbial cells containing nitrilase activity are useful inthis invention. Examples of such a transformed microbial cell are E.coli SS1001 (ATCC PTA-1177) and E. coli SW91 (ATCC PTA-1175) whichcontain A. facilis 72W nitrilase activity. The inorganic salt(s) ispreferably ammonium carbamate. The conditions for the method includewhere the pH of the aqueous suspension of microbial cells havingnitrilase activity is from about pH 6 to about pH 10 and the temperatureof the aqueous suspension of microbial cells ranges from about 0° C. toabout 45° C. The microbial cells are preferably immobilized inpolyacrylamide gel, alginate, or carrageenan. In a preferred embodiment,the microbial cells are immobilized in carrageenan, the inorganicbicarbonate salt is selected from the group consisting of ammoniumbicarbonate and potassium bicarbonate, and the resulting totalconcentration of the inorganic bicarbonate salt(s) in the aqueoussuspension ranges from about 100 mM to about 1000 mM. In anotherpreferred embodiment of the method, the microbial cells areunimmobilized, the compound is sodium bicarbonate, and the resultingtotal concentration of sodium bicarbonate in the aqueous suspensionranges from about 100 mM to about 1000 mM.

[0010] In another preferred embodiment, the microbial cells areimmobilized in alginate, the compound added to the aqueous suspension isan inorganic carbamate salt, preferably ammonium carbamate, and theprocess includes the additional step of adding a calcium salt that doesnot adversely affect enzyme activity or stability (preferably in theform of calcium acetate or calcium chloride) to the aqueous suspensionof at least 2 mM, and preferably in the range of from about 2 mM toabout 5 mM.

BRIEF DESCRIPTION OF THE BIOLOGICAL DEPOSITS

[0011] Applicants have made the following biological deposits under theterms of the Budapest Treaty: Depositor Identification Int'l. DepositoryReference Designation Date of Deposit Acidovorax facilis 72-PF-17 ATCC55745 8 March 1996 Acidovorax facilis 72W ATCC 55746 8 March 1996Acidovorax facilis 72-PF-15 ATCC 55747 8 March 1996 E. coli SS1001 ATCCPTA-1177 11 January 2000 E. coli SW91 ATCC PTA-1175 11 January 2000

[0012] As used herein, “ATCC” refers to the American Type CultureCollection international depository located at 10801 UniversityBoulevard, Manassas, Va. 20110-2209, U.S.A. The “Int'l. DepositoryDesignation” is the accession number to cultures on deposit with theATCC.

[0013] The listed deposits will be maintained in the indicatedinternational depository for at least thirty (30) years and will be madeavailable to the public upon the grant of a patent disclosing it. Theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentaction.

DETAILED DESCRIPTION OF THE INVENTION

[0014] A method for stabilizing the nitrilase activity of unimmobilizedor immobilized microbial cells has been developed, wherein the cells arestored in an aqueous buffer containing from about 0.100 M to thesaturation concentration of at least one inorganic salt of bicarbonate,carbonatem, or carbamate, including but not limited to ammonium, sodium,and potassium salts of bicarbonate, carbonate, or carbamate. Storagesolutions containing at least 100 mM bicarbonate, carbonate, orcarbamate salt significantly limit microbial contamination orputrefaction of the stored enzyme catalyst, and also stabilize thedesired nitrilase activity of the unimmobilized or immobilized cells.Microorganisms which are characterized by a nitrilase activity and arestably preserved by this method include but are not limited toAcidovorax facilis 72-PF-15 (ATCC 55747), Acidovorax facilis 72-PF-17(ATCC 55745), and Acidovorax facilis 72W (ATCC 55746) and microorganismstransformed so as to include nitrilase activity (for instance, E. coliSS1001 (ATCC PTA-1177) or E. coli SW91 (ATTC PTA-1175), each transformedwith the nitrilase of Acidovorax facilis 72W).

[0015] In this disclosure, a number or terms and abbreviations are used.The following definitions are provided.

[0016] “High pressure liquid chromatography” is abbreviated HPLC.

[0017] “Polyacrylamide gel” is abbreviated PAG.

[0018] The term “nitrilase” refers to an enzyme that can be used todirectly convert a nitrile to the corresponding carboxylic acid inaqueous solution without the intermediate formation of an amide(R—CN+2H₂O→RCOOH +NH₃).

[0019] The term “stabilize” refers to maintaining the activity of anenzyme catalyst in whole unimmobilized or immobilized microbial cellssuch that there is no continuous loss of enzyme activity over timerelative to the initial enzyme catalyst activity. The effect is tomaintain enzyme activity at levels similar to those observed at Day 0following preparation of the enzyme catalyst such that the useful lifeof the enzyme catalyst is prolonged.

[0020] The terms “preserve” or “preservation” refer to preventing orlimiting putrefaction (microbial contamination or cell lysis) ofnitrilase-containing microbial cells. The effect is to significantlylimit contamination of the unimmobilized or immobilized cell aqueoussuspension by maintaining cell integrity without lysis relative to cellsnot treated with this method.

[0021] U.S. Pat. No. 5,814,508 and U.S. Pat. No. 5,858,736 describeprocesses for the preparation of five-membered ring lactams orsix-membered ring lactams from aliphatic α,ω-dinitriles, where analiphatic α,ω-dinitrile is first converted to an ω-cyanocarboxylic acidammonium salt in aqueous solution using microbial cells having analiphatic nitrilase (EC 3.5.5.7) activity, followed by the directconversion of the ammonium salt of the ω-cyanocarboxylic acid to thecorresponding lactam by hydrogenation in aqueous solution. In the courseof preparing and storing unimmobilized or immobilized cells havingnitrilase activity for this process as suspensions in aqueous solutioncontaining, for example, phosphate buffer or physiological saline, itwas found that many of these solutions did not prevent microbialcontamination or putrefaction of the stored catalyst at temperaturesranging from approximately 5° C. to 30° C. In those cases wheremicrobial contamination or putrefaction was not observed, a significantloss of nitrilase activity still occurred during storage. Freezing ofimmobilized cell catalysts typically resulted in the destruction of theimmobilization matrix, and while unimmobilized cells could be storedfrozen without significant loss of activity for months, a significantcost was associated with this method. Because storage of these microbialcatalysts for weeks or months before use after preparation without lossof enzyme activity or putrefaction is desirable, a method of stablypreserving aqueous suspension of the unimmobilized or immobilizedmicrobial cell catalysts in storage was sought.

[0022] An examination of the effect of various inorganic salts on thestorage stability of the nitrilase activity of unimmobilized orimmobilized Acidovorax facilis 72W (ATCC 55746), including some of thosedescribed in EP 0707061 for the stable preservation of the nitrilaseactivity of Gordona terrae MA-1, was conducted. Storage solutionscontaining potassium phosphate, ammonium sulfate, or ammonium chlorideat various concentrations did not prevent the loss of nitrilase activitywith time (see accompanying examples). Salts of organic acids such asacetate or propionate, previously reported to stabilize the nitrilehydratase activity of microbial cells, were also examined, even thoughthe nitrile hydratase and nitrilase enzymes are different types ofenzymes with different mechanisms for catalyzing nitrile hydrolysis toamides or acids, respectively. As disclosed in the accompanyingexamples, ammonium acetate or ammonium propionate were not effective inpreserving the nitrilase activity of unimmobilized or immobilizedAcidovorax facilis 72W cells.

[0023] It was discovered that carbonate, bicarbonate, and carbamatesalts, including but not limited to ammonium bicarbonate, sodiumbicarbonate, potassium bicarbonate, and ammonium carbamate, areeffective for both 1) preventing significant microbial contamination orputrefaction of suspensions of unimmobilized or immobilized cellscontaining nitrilase activity (specifically, Acidovorax facilis 72Wcells and including microbes transformed to possess the nitrilaseactivity, for instance, of A. facilis 72W), and 2) stabilizing thenitrilase activity of these suspensions, as demonstrated in theaccompanying examples.

[0024] Organisms Potentially Expressing Nitrilase Genes:

[0025] Many different genera are known to express nitrilase activity orcontain genes that are thought to express nitrilase proteins. Both Gramnegative (e.g., Comamonas, Acidovorax, Klebsiella (McBride et al., Appl.Environ. Microbiol. 52:325-330 (1986)), Acinetobacter (Yamamoto et al.,Agric. Biol. Chem. 55:1459-1466 (1991)), Alcaligenes (Yamamoto et al.,J. Ferm. Bioeng. 73:425-430 (1992)) and Gram positive (e.g., Rhodococcus(Bhalla et al., Acta. Biotechnol. 15:297-306 (1995)), Arthrobacter(Bandyopadhyay et al., Appl. Environ. Microbiol. 51:302-306 (1986)),Gordona (DNA encoding novel nitrilase from Gordona terrae., NCBI GenBankAccession Number E12616, Patent: JP1997037788-A 1 10-February, 1997),Bacillus (Cramp et al., Microbiol. 143:2313-2320 (1997)) bacteriaexpressing nitrilase activity are known. A number of reports are alsoknown from plants (Nicotiana tabacum (Tsunoda, NCBI GenBank AccessionNumber D83078); Arabidopsis thaliana (Zhou et al., Plant Physiol. 110,1048 (1996)), and fungi (e.g., Fusarium solani (Harper, Biochem. J.167:685-692 (1977)).

[0026] In higher organisms (Drosophila, Caenorhabditis, Homo sapiens),genes have been found that appear to code for nitrilase proteins(Pekarsky et al., Proc. Nat. Acad. Sci. U.S.A. 95(15):8744-8749 (1998)).No discrete function has been reported for these genes. However, theyretain high homology to known nitrilase genes and are expected to showtypical nitrile hydrating activity. Nitrilase genes so far sequencedretain a common motif at the active site (Novo et al., FEBS Lett.367:275-279 (1995)) and thus are expected to be susceptible topreservation by the present invention.

[0027] Transformation and Expression of Nitrilase Genes:

[0028] Heterologous expression of nitrilase activity from transformedhosts has been reported (Kobayashi et al., Proc. Nat. Acad. Sci. 90:247(1993); Kobayashi et al., J. Biol. Chem. 267:20746 (1992); Kobayashi etal., Biochem. 31:9000-9007 (1992); U.S. Pat. No. 4,810,648). In somecases the hosts must be specifically modified in order to express goodenzyme activity (Levy-Schil et al., Gene 161:15-20 (1995); U.S. Pat. No.5,830,693; U.S. Pat. No. 5,635,391). In cases where good nitrilaseactivity is found, the enzyme expressed by the transformed host retainsthe nitrile hydration activity of the native enzyme. The presentinvention will preserve such nitrile hydration activity expressed bytransformed hosts, as evidenced by the data in Example 11 whereAcidovorax facilis 72W nitrilase was transformed into in E. coli.

[0029] Heterologous Host Cells:

[0030] The active Acidovorax facilis 72W nitrilase protein may beproduced in heterologous host cells, preferably in microbial hosts.Particularly useful in the present invention will be cells that arereadily adaptable to large-scale fermentation methods. Such organismsare well known in the art of industrial bioprocessing, examples of whichmay be found in “Recombinant Microbes for Industrial and AgriculturalApplications”, Murooka et al., eds., Marcel Dekker, Inc., New York, N.Y.(1994), and include fermentative bacteria as well as yeast andfilamentous fungi. Host cells may include but are not limited toComamonas sp., Corynebacterium sp., Brevibacterium sp., Rhodococcus sp.,Azotobacter sp., Citrobacter sp., Enterobacter sp., Clostridium sp.,Klebsiella sp., Salmonella sp., Lactobacillus sp., Aspergillus sp.,Saccharomyces sp., Zygosaccharomyces sp., Pichia sp., Kluyveromyces sp.,Candida sp., Hansenula sp., Dunaliella sp., Debaryomyces sp., Mucor sp.,Torulopsis sp., Methylobacteria sp., Bacillus sp., Escherichia sp.,Pseudomonas sp., Rhizobium sp., and Streptomyces sp. Particularlypreferred is E. coli. Microbial expression systems and expressionvectors containing regulatory sequences that direct high levelexpression of foreign proteins are well known to those skilled in theart.

[0031] Particular Strains:

[0032]Acidovorax facilis 72W (ATCC 55746) was isolated from soilcollected in Orange, Tex. Standard enrichment procedures were used withthe following medium (E2 Basal Medium, pH 7.2): E2 Basal Medium g/LKH₂PO₄ 1.4 NaH₂PO₄ 0.69 sodium citrate 0.1 CaCl₂.2H₂O 0.025 KCl 0.5 NaCl1.0 MgSO₄.7H₂O 0.5 FeSO₄.7H₂O 0.05 CoCl₂.6H₂O 0.01 MnCl₂.4H₂O 0.001ZnCl2 0.0005 NaMoO₄.2H₂O 0.0025 NiCl₂.6H₂O 0.01 CuSO₄.2H₂O 0.005 biotin0.0002 folic acid 0.0002 pyridoxine.HCl 0.001 riboflavin 0.0005thiamine.HCl 0.00005 nicotinic acid 0.0005 pantothenic acid 0.0005vitamin B₁₂ 0.00001 p-aminobenzoic acid 0.0005

[0033] The following supplementations were made to the E2 basal mediumfor the enrichments described above: Strain Enrichment Nitrogen SourceOther Supplements A. facilis 72W 0.2% (v/v) ethylsuccinonitrile 0.3%(v/v) glycerol

[0034] Strains were originally selected based on growth and ammoniaproduction on the enrichment nitrile. Isolates were purified by repeatedpassing on Bacto® Brain Heart Infusion Agar (DIFCO, Detroit, Mich.)followed by screening for ammonia production from the enrichmentnitrile. Purified strains were identified based on their carbon sourceutilization profile on a Biolog® test system (Hayward, Calif.) usingGram negative test plates.

[0035] Nitrilase Activity:

[0036] For testing nitrilase activity, E2 basal medium with 10 g/Lglucose was used to grow Acidovorax facilis 72W. The medium wassupplemented with 25 mM (±)-2-methylglutaronitrile. A 10 mL volume ofsupplemented E2 medium was inoculated with 0.1 mL of frozen stockculture. Following overnight growth at room temperature (22-25° C.) on ashaker at 250 rpm, the 10 mL inoculum was added to 990 mL of freshmedium in a 2 L flask. The cells were grown overnight at roomtemperature with stirring at a rate high enough to cause bubbleformation in the medium. Cells were harvested by centrifugation, washedonce with 50 mM phosphate buffer (pH 7.2)/15% glycerol and theconcentrated cell paste was immediately frozen on dry ice and stored at−65° C. Adiponitrile, 10 mM, was also used in the 1 liter fermentations.Fermentations were stopped after 16-20 hours of growth. The cellsuspension was chilled to 4° C., harvested by centrifugation and frozenat −60° C. following one wash with 15% glycerol in 0.05 M phosphatebuffer (pH 7.2).

[0037] Thawed cell pastes were used for testing nitrilase activity. Thedesired property of the microorganism is a nitrile hydrolyzing activitycapable of regiospecific hydration of a dinitrile compound in theabsence of interfering nitrile hydratase and amidase activities.Microorganisms tend to undergo mutation. Some mutations may be favorableto the desired nitrile conversion. Thus, even mutants of the nativestrain may be used to carry out the process of the instant invention.

[0038] The present invention is not limited to the particular organismsmentioned above, but includes the use of variants and mutants thereofthat retain the desired property. Such variants and mutants can beproduced from parent strains by various known means such as x-rayradiation, UV-radiation, and chemical mutagens.

[0039] Mutants of Acidovorax facilis 72W (ATCC 55746) with reducedcapacity to produce the undesirable 2-methylglutaric acid by-productduring hydrolysis of 2-methylglutaronitrile to 4-cyanopentanoic acidwere selected based on their inability to use 2-methylglutaronitrile asa carbon and energy source. Specifically, an overnight culture of strainAcidovorax facilis 72W grown on LB/succinate medium (1% (w/v)Bacto-tryptone (DIFCO, Detroit, Mich.), 0.5% (w/v) Bacto-yeast extract(DIFCO), 1% (w/v) NaCl, 0.5% (w/v) sodium succinate hexahydrate) wasexposed to 100 μg/mL solution of N-methyl-N′-nitro-N-nitrosoguanidine, amutagenic agent, for approximately 30 minutes. This resulted in a 99.9%reduction in viable cells in the culture. Mutagenized cells were washedfree of the mutagen by centrifugation in sterile, 1 M sodium phosphatebuffer (pH 7.2). Washed cells were resuspended in LB/succinate mediumand grown overnight at 30° C. Cells were then washed by centrifugationin sterile, 50 mM sodium phosphate buffer (pH 7.2) and resuspended in E2minimal medium (without glucose) containing 0.2% (v/v)2-methylglutaronitrile, and the antibiotics cycloserine, 0.2 mg/mL andpiperacillin, 40 μg/mL. Cells were incubated overnight at 30° C. andagain washed in sterile, 50 mM sodium phosphate buffer (pH 7.2). Washedcells were spread on agar plates containing a non-selective medium: E2minimal medium (without glucose) plus 0.2% (v/v) 2-methylglutaronitrileand 0.5% (w/v) sodium succinate hexahydrate, at a concentration of40-100 colony-forming units per plate. Plates were incubated forapproximately 48 h at 30° C. to allow colonies to develop. Colonieswhich developed were replica plated onto agar plates containingselective medium: E2 minimal medium (without glucose) plus 0.2% (v/v)2-methylglutaronitrile. Plates were incubated 48 h at 30° C. to allowcolonies to develop. Mutants with desirable qualities do not grow wellon the selective medium. Therefore, after 48 h, replicated plates werecompared and strains showing growth only on non-selective medium weresaved for further testing.

[0040] In total, approximately 5,120 colonies were checked from 89plates and 19 strains with the desirable qualities were identified.These mutant strains were further tested for growth in liquid, E2minimal medium (without glucose) plus 0.2% (v/v) 2-methylglutaronitrile.Strains which showed little or no growth in this medium were screenedfor their ability to produce 2-methylglutaric acid during growth inliquid medium consisting of E2 minimal medium (without glucose) plus0.2% (v/v) 2-methylglutaronitrile and 0.5% (w/v) sodium succinatehexahydrate. As a result of this process, two mutant strains, identifiedas Acidovorax facilis 72-PF-15 (ATCC 55747) and Acidovorax facilis72-PF17 (ATCC 55745) were chosen for further development due to theirgreatly diminished capacity to produce 2-methylglutaric acid.

[0041] To produce biocatalyst for stabilization studies, the followingmedia can be used. Strain Medium 72-PF-15 Lauria-Bertani Medium(Bacto ®tryptone, 10 g/L + Bacto ® yeast extract, 5 g/L + NaCl, 10 g/L) + 0.5%(w/v) sodium succinate.6H₂O 72 W E2 + 1% (w/v) glucose + 0.4% (w/v)adipamide

[0042] 0.1 mL of frozen stock culture. Following overnight growth at 28°C. with shaking at 250 rpm, the growing cell suspension was transferredto 1 L of the same medium in a 2 L flask and growth continued at 28° C.with shaking. The 1 L growing cell suspension was then added to 9 L ofthe same medium in a 10 L fermentation vessel where growth continued.Nominal conditions in the fermenter were: ≧80% oxygen saturation, 25°C., pH 7.2 and 300-1000 rpm. After 20-91 hours, the vessel was chilledto 8-12° C. and cells harvested by centrifugation. The concentrated cellpaste was immediately frozen on dry ice and stored at −70° C. until use.Numerous other supplementations which will serve as carbon and nitrogensources for cell growth in E2 basal medium are known to those skilled inthe art. These, as well as complex nutrient media, can be used toproduce biocatalyst. The particular media described above should not beviewed as restrictive.

[0043] Cells having nitrilase activity and which are collected fromculture broth for storage in cell suspensions, or for use in thepreparation of immobilized cells which are subsequently stored insuspension, can be used directly without washing prior to storage or usein immobilization, or can be washed with the storage buffer prior tostorage or immobilization. Cells may also be collected from culturebroth and frozen prior to storage in cell suspensions or as suspensionsof immobilized cells.

[0044] Nitrilase Assay:

[0045] Nitrilase activity of unimmobilized cells was determined eitherby benzonitrile conversion to benzoic acid as measuredspectrophotometrically by the increase in absorbance at 245 nm, or by2-methyglutaronitrile conversion to 4-cyanopentanoic acid ammonium saltmeasured by HPLC. For the benzonitrile-based nitrilase assay, 5 mgfrozen wet cell paste/mL was suspended in 0.1 M phosphate buffer (pH7.0). Benzonitrile was added to a final concentration of 1 mM. Thesuspension was shaken at 250 rpm at 30° C. and samples were collectedinto a 4x volume of 0.04 M phosphoric acid stop solution at 0.5, 1, 2and 4 minutes. After five minutes centrifugation at 15,800 x g, theresulting supernatant was analyzed spectrophotometrically. Based on anempirically determined extinction coefficient for benzonitrile andbenzoic acid in buffer plus stop solution, unit activity for thenitrilase was calculated by the following equation, where x is cell pathlength in centimeters (with correction for dilutions):

ΔC (Molar)=ΔO.D. ₂₄₅/1.81 Molar⁻¹ cm⁻¹(x)

[0046] For the 2-methylglutaronitrile-based assay, 1 mL of 50 mg drycell weight/mL cell suspension (approximately 200 mg frozen cellpaste/mL) in 0.100 M KH₂PO₄ (pH 7.0) buffer was added to 3.0 mL of 0.40M 2-methylglutaronitrile in deionized water at 25° C. The reactionmixture was maintained at 25° C. with stirring, and 0.180 mL aliquotswere removed and mixed with 5 μL of 6.0 N HCl and 20 μL of externalstandard solution (0.75 M N-methylpropionamide) in deionized water. Theresulting mixture was immediately mixed, then centrifuged for 2 min at12,000 rpm. The supernatant was analyzed by HPLC using a refractiveindex detector and a Supelcosil LC-18-DB column (25 cm×4.6 mm diameter),using an aqueous solvent containing 10 mM sodium acetate, 10 mM aceticacid and 7.5% methanol.

[0047] The nitrilase activity of immobilized cells was determined bymixing 16.5 g of an immobilized cells catalyst with 10.8 g of2-methyglutaronitrile and 72.1 mL of 20 mM potassium phosphate buffer(pH 7.5) at 25° C. Aliquots (0.180 mL) were removed and mixed with 5 μLof 6.0 N HCl and 20 μL of external standard solution (0.75 MN-methylpropionamide) in deionized water. The resulting solution wasimmediately mixed, then centrifuged for 2 min at 12,000 rpm and thesupernatant analyzed for 4-cyanopentanoic acid ammonium salt by HPLC asdescribed above.

[0048] Immobilization:

[0049] Nitrilase-containing cells can be immobilized using any of themethods and procedures known to those skilled in the art. These methodsinclude, but are not limited to, immobilization in alginate,carrageenan, polyvinylalcohol, or polyacylamide gel, as well asimmobilization by adsorption or attachment to ion exchange resins,diatomaceous earth (celite), activated carbon, silica, porous glassbeads, alumina, zirconia, titania and the like.

[0050] Stabilization Compounds:

[0051] The compound or compounds used in the present invention tostabilize nitrilase activity and preserve the integrity (prevent themicrobial contamination or lysis) of unimmobilized or immobilized cellsare selected from the group consisting of inorganic salts of carbonate,bicarbonate, and carbamate. Types of inorganic salts include, but arenot limited to, sodium salts, potassium salts, and ammonium salts. Asingle compound selected from this group may be used as a nitrilasestabilizer, or two or more compounds selected from this group may beincluded together in the preservative solution. Solutions containingcarbonate or bicarbonate salts may additionally be prepared by sparginga buffered solution with carbon dioxide to generate the desired salt insitu, with appropriate pH adjustment. Solutions containing a carbamatesalt may additionally be prepared by generating carbamic acid in water,followed by the preparation of the desired salt in situ, withappropriate pH adjustment.

[0052] In the case of carrageenan-immobilized cells, ammonium orpotassium salts are preferred, as these cations beneficially increasethe degree of crosslinking in the carrageenan gel.

[0053] In the case of alginate-immobilized cells, the optional additionof up to 5 mM calcium ion (e.g., any salt of calcium ion that does notadversely affect enzyme activity or stability, preferably in the form ofcalcium acetate or calcium chloride) to the bicarbonate, carbonate, orcarbamate salts is preferred, as calcium ions beneficially increase thedegree of crosslinking in the alginate gel. When carbonate orbicarbonate salts are used to stabilize the activity ofalginate-immobilized cells, the addition of calcium ion is preferably inthe range of about 2 mM up to about 5 mM. Above a concentration of about5 mM calcium ion, a precipitate of highly-insoluble calcium carbonate isproduced from the reaction of calcium ion with the carbonate ion presentin either carbonate or bicarbonate solutions. The concentration ofcalcium ion is at least 2 mM, and preferably from 2 mM up to about 5 mM.The reaction of calcium ion with carbonate ions to form calciumcarbonate may adversely affect the desired increase in crosslinking ofcalcium-alginate gels by calcium ion.

[0054] The use of inorganic salts of carbamate is preferred to preservenitrilase activity of alginate-immobilized cells, because there is nosignificant reaction of calcium ion with carbamate to produce aninsoluble calcium salt; for example, mixing equal volumes of 0.20 Mcalcium acetate and 0.20 M ammonium carbamate at pH 7.3 produces noprecipitate in the resulting solution which contains 0.10 Mconcentrations of both calcium and carbamate ions. The optional additionof greater than 5 mM calcium ion to carbamate salts may be used tofurther beneficially increase the degree of crosslinking in the alginategel containing the immobilized cells.

[0055] In addition to stabilizing the nitrilase activity ofunimmobilized or immobilized cells, the use of carbonate, bicarbonate,or carbamate salt also prevents the microbial contamination orputrefaction of suspensions of the unimmobilized or immobilized cells.

[0056] The use of glutaraldehyde and polyethylenimine for the chemicalcrosslinking of unimmobilized microbial cells (U.S. Pat. No. 4,288,552and U.S. Pat. No. 4,355,105) and immobilized microbial cells (Takata etal., Bioprocess Technol. 16:53-65 (1993); Tosa et al., Biotechnol.Bioeng. 21:1697-1709 (1979); Bahulekar et al., Enzyme Microb. Technol.13:858-868 (1991); Chao et al., Biotechnol. Bioeng. 28:1289-1293 (1986))has been previously reported to significantly stabilize the enzymaticactivity of these catalysts under reaction conditions in manyapplications, including the production of high concentrations ofcarboxylic acid salts. Chemical crosslinking of unimmobilized cells orimmobilized cells and subsequent storage in aqueous buffers that do notcontain carbonate, bicarbonate, or carbamate salts has not been found toprevent the loss of nitrilase activity over time. Carbonate,bicarbonate, or carbamate salts may be also be used to stabilizenitrilase activity and preserve the integrity (prevent the microbialcontamination or lysis) of unimmobilized or immobilized cells which havebeen chemically-crosslinked with glutaraldehyde and polyethylenimine.

[0057] The concentration of the inorganic carbonate, bicarbonate, orcarbamate salt(s) in the aqueous preservative solution may range fromabout 0.10 M to the saturation concentration of said inorganic salts.The saturation concentration of each salt will vary with temperature andconcentration, and for stabilizing immobilized catalyst, is preferablyin the range of from 0.25 M to 1.0 M. In the case of unimmobilizedcells, it has been observed that at concentrations of bicarbonate saltsequal to or greater than 0.50 M, the cell suspensions becomeheterogenous, although no loss of nitrilase activity is observed;therefore the preferable range of concentration of this inorganic saltfor the stabilization of unimmobilized cells is from 0.10 M to 0.50 M.

[0058] The pH of the suspension is preferably from 5 to 10, with a morepreferred pH range of from 6.5 to 8.0. The pH of the aqueous storagesuspensions may be adjusted with a suitable acid, such as acetic,hydrochloric, sulfuric, or phosphoric acid, or a suitable base, such asammonium hydroxide, sodium hydroxide, or potassium hydroxide.

[0059] The temperature at which the unimmobilized or immobilized cellsare stably preserved in aqueous solutions containing one or moreinorganic salts of carbonate, bicarbonate, or carbamate may range fromapproximately 0° C. (or just above the freezing point of thepreservative solution) and 45° C., with a preferred range of from 5° C.to 30° C.

[0060] The present invention is further defined in the followingExamples. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions.

EXAMPLES

[0061] Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,DC. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents and materials used for the growthand maintenance of bacterial cells were obtained from Aldrich Chemicals(Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.), GIBCO/BRL(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.) unlessotherwise specified.

[0062] The meaning of abbreviations is as follows: “sec” meanssecond(s), “min”means minute(s), “h” means hour(s), “d” means day(s),“μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s),“mM” means millimolar, “M” means molar, “mmol” means millimole(s).

Example 1 Storage Stability of Unimmobilized Acidovorax facilis CellNitrilase Activity

[0063]Acidovorax facilis 72W cells were isolated from cultured broth bycentrifugation, and then suspended at a concentration of 1-5% dry cellweight in aqueous potassium phosphate (0.10 M or 1.00 M), sodium acetate(0.10 M or 1.00 M), or sodium bicarbonate (0.10 M or 0.30 M) at pH 7.3,and the resulting suspensions stored at 5° C. in capped glass bottles.Samples were removed from the cell suspensions over time and the samplesassayed for nitrilase activity. Table 1 shows the relative nitrilaseactivity of the stored cell suspensions, with the nitrilase activity ofthe suspensions at day 0 defined as 100%. Cells stored for 92 days in0.10 M or 0.30 M sodium bicarbonate retained a significantly-higherpercentage of the initial nitrilase activity when compared to storage ineither 0.10 M or 1.0 M sodium acetate or potassium phosphate. TABLE 1nitrilase activity (%) KH₂PO₄ NaOAc NaHCO₃ Day 0.10 M 1.00 M 0.10 M 1.00M 0.10 M 0.30 M 0 100 100 100 100 100 100 39 93 86 87 82 97 — 41 — — — —— 106 64 96 86 72 82 104 — 69 — — — — — 92 92 81 78 63 76 95 95

Example 2 Immobilization of Acidovorax facilis 72W in CarrageenanParticles

[0064]Acidovorax facilis 72W wet cell paste (45 grams) was mixed with 45mL of 0.88% NaCl, and the resulting suspension heated at 50° C. for 1 hto inactivate undesirable nitrile hydratase activity. The 50° C. cellsuspension was then added to 135.0 g of a 5 wt % solution of PronovaISAGEL RG300 carrageenan at 55° C. with mixing. The resulting suspensionwas then immediately gelled by cooling to 5° C. in an ice/water bath for1 h. The resulting gel was cut into particles approximately 2 mm indiameter, and the immobilized cell particles then hardened in 450 mL of0.30 M KCl, 20 mM KH₂PO₄ (adjusted to pH 7.0 with KOH) at 5° C. for 18h. The hardening solution was then removed by washing the immobilizedcell particles three times with 5 mM KCl, 20 mM KH₂PO₄ (pH 7.0) at 5° C.

Example 3 Immobilization of Acidovorax facilis 72W in Polyacrylamide GelParticles

[0065] A solution of 13.8 g acrylamide and 1.20 g methylenebisacrylamidein 15.0 mL water at 25° C. was added with stirring to a suspension of30.0 g (wet cell weight) Acidovorax facilis 72W in 82 mL of 0.10 MKH₂PO₄ (pH 7.0) at 10° C. with stirring. To the resulting mixture wasadded tetramethylethylenediamine (0.45 mL) and 7.5 mL of 5% (w/v)potassium persulfate at 25° C. The resulting polymerized gel was storedfor 1 h at 10° C. in an ice bath, then cut into particles approximately2 mm in diameter. The immobilized cell particles were washed twice with50 mM KH₂PO₄ (pH 7.0) at 5° C.

Example 4 Storage Stability of Immobilized Acidovorax facilis NitrilaseActivity

[0066]Acidovorax facilis 72W cells (5% dry cell weight) were immobilizedin Pronova ISAGEL RG300 carrageenan (3.0 wt %) as in Example 2 above andthe resulting catalyst particles suspended in aqueous 1.0 M potassiumphosphate, ammonium acetate, ammonium propionate, ammonium bicarbonate,or ammonium sulfate, or in 50 mM potassium phosphate containing 1.0 Mammonium chloride at pH 7.3. The resulting immobilized catalystsuspensions were stored at 5° C. in capped glass bottles, and sampleswere removed over time and the immobilized Acidovorax facilis 72W cellsassayed for nitrilase activity.

[0067] Table 2 shows the relative nitrilase activity of the storedimmobilized cell suspensions, with the activity at day 0 defined as100%. Immobilized cells stored in 1.0 M ammonium bicarbonate retained asignificantly-higher percentage of the initial nitrilase activity whencompared to storage in 1.0 M potassium phosphate, ammonium acetate,ammonium propionate, or in 50 mM potassium phosphate containing 1.0 Mammonium chloride, at pH 7.3 and 5° C. TABLE 2 nitrilase activity (%)NH₄CL ammonium (1.0 M) NH₄HCO₃ KH₂PO₄ NH₄OAc propionate KH₂PO₄ (NH₄)₂SO₄day (1.0 M) (1.0 M) (1.0 M) (1.0 M) (50 mM) (1.0 M)  0 100 100 100 100100 100 15 — — — — — 93 23 96 71 78 84 83 — 27 — — — — — 72 43 — 53 — 79— — 45 98 — 67 — — — 46 — — — — 67 — 56 102 — 60 — — — 60 — — — — 62 —74 — — — 73 — — 81 99 — 50 — — — 95 — — 40 — — — 98 107 — — 66 — —

Example 5 Storage Stability of Immobilized Acidovorax facilis NitrilaseActivity

[0068]Acidovorax facilis 72W cells (5% dry cell weight) were immobilizedin Pronova ISAGEL RG300 carrageenan (3.0 wt %) as in Example 2 above andthe resulting catalyst particles suspended in aqueous 1.0 M ammoniumbicarbonate at pH 7.3. The resulting immobilized catalyst suspension wasstored at 25° C. in a capped glass bottle, and samples were removed overtime and the immobilized Acidovorax facilis 72W cells assayed fornitrilase activity.

[0069] Table 3 shows the relative nitrilase activity of the storedimmobilized cell suspension, with the activity at day 0 defined as 100%.TABLE 3 nitrilase activity (%) day NH₄HCO₃ 0 100 10 94 23 94 38 92 59 9378 88 105 91

Example 6 Microbial Contamination in Suspensions of ImmobilizedAcidovorax facilis

[0070]Acidovorax facilis 72W cells (5% dry cell weight) were immobilizedin Pronova ISAGEL RG300 carrageenan (3.0 wt %) as in Example 2 above orin polyacrylamide gel (PAG, 10 wt %) as in Example 3 above, and theresulting catalyst particles suspended in aqueous solutions containing:a) 50 mM potassium phosphate (pH 7.0); b) 1.0 M ammonium acetate (pH7.3); c) 1.0 M ammonium bicarbonate (pH 7.3); or d) 50 mM potassiumphosphate/1.0 M ammonium chloride (pH 7.0). The resulting immobilizedcatalyst suspensions were stored at 5° C. in capped glass bottles, andsamples of the storage solution were removed over time. The samples wereexamined for microbial contamination by measurement of the change inoptical density (OD) at 600 nm.

[0071] Example 4, Table 2, shows that immobilized cells stored in 1.0 Mammonium bicarbonate retained a significantly-higher percentage of theinitial nitrilase activity when compared to storage in 1.0 M potassiumphosphate, ammonium acetate, ammonium propionate, or in 50 mM potassiumphosphate containing 1.0 M ammonium chloride, at 5° C. Microbialcontamination, indicated by an increase in the measured optical density(OD) of the storage buffer over time, occurred with cells immobilized ineither PAG or RG300 carrageenan when stored in 50 mM potassiumphosphate, while no significant microbial contamination or putrificationwas observed for cells stored in 1.0 M ammonium acetate, ammoniumbicarbonate, or 1.0 M ammonium chloride/50 mM potassium phosphate (Table4). Of the three storage suspensions which showed no microbialcontamination or putrefaction, only the immobilized cells stored in 1.0M ammonium bicarbonate showed no significant loss of nitrilase activity(see Example 4, Table 2). TABLE 4 OD (600 nm) RG300, PAG, RG300, RG300,RG300, NH₄Cl (1.0 M) KH₂PO₄ KH₂PO₄ NH₄OAc NH₄HCO₃ KH₂PO₄ day (50 mM) (50Mm) (1.0 M) (1.0 M) (50 mM) 1 — 0.026 0.055 0.045 0.054 2 0.061 — — — —5 0.047 0.075 — — 0.035 8 0.079 0.137 — — 0.017 11 0.207 — — — — 12 —0.115 0.008 0.001 0.040 15 0.360 — — — — 16 — 0.276 — — 0.067 18 0.419 —— — — 19 — 0.221 — — 0.033 23 — 0.521 — — 0.050 26 — — 0.029 0.073 — 33— — 0.027 0.082 — 46 — — 0.048 0.104 — 47 — — — — 0.032 57 — — 0.0850.072 — 61 — — — — 0.033

Example 7 Immobilization of Acidovorax facilis 72W in Carrageenan BeadsWith Glutaraldehyde/Polyethylenimine-Crosslinking

[0072] Into a 250 mL media bottle equipped with magnetic stir bar andcontaining 64 g of water at 50° C. was slowly added 3.38 g ofcarrageenan with rapid stirring. The mixture was heated to 75-80° C.with rapid stirring until the carrageenan was completely dissolved, andthe resulting solution cooled to 55-56° C. (gelling temperature ca. 52°C.) in a thermostated water bath. A suspension of 23.4 g of A. facilis72W cells (wet cell weight) in 21.56 g of 0.30 M sodium bicarbonatebuffer (pH 7.3) was heated to 50° C. for 1 hour to deactivate nitrilehydratase. The cell suspension at 50° C. was then added to thecarrageenan solution at 55-56 C. with stirring, then thecell/carrageenan mixture was immediately added slowly to 450 mL ofsoybean oil at 50° C. with stirring using an overhead stirrer. Aftercell/carrageenan droplets of the desired size were produced in the oilby controlling the stirring rate, the temperature of the oil was reducedto 35° C. to gel the droplets, and the oil decanted from the resultingbeads. The beads were washed with 0.3 M potassium chloride in 50 mMpotassium phosphate (pH 7.0), then resuspended in 250 mL of this samebuffer, and 2.0 g of 25 wt % glutaraldehyde in water was added and thebeads mixed for 0.5 h at 25° C. To the mixture was then added 9.0 g of11 wt % polyethylenimine (BASF Lupasol PR971L, average Mw ca. 750,000)in water, and the beads mixed for an additional hour at 25° C. The beadswere then washed with 0.3 M potassium chloride in 50 mM potassiumphosphate (pH 7.0) and stored in this same buffer for at least 18 h at5° C. to further harden the beads.

Example 8 Storage Stability ofGlutaraldehyde/Polyethlenimine-Crosslinked, Immobilized Acidovoraxfacilis 72W Nitrilase Activity

[0073]Acidovorax facilis 72W cells (5% dry cell weight) were immobilizedin Pronova ISAGEL RG300 carrageenan (3.0 wt %) as described in Example7, and the resulting catalyst beads suspended in aqueous 1.0 M ammoniumbicarbonate at pH 7.3. The resulting immobilized catalyst suspension wasstored at 5° C. in a capped glass bottle, and samples were removed overtime and the immobilized Acidovorax facilis 72W cells assayed fornitrilase activity.

[0074] Table 5 shows the relative nitrilase activity of the storedimmobilized cell suspension, with the activity at day 0 defined as 100%.TABLE 5 day nitrilase activity (%) 0 100 61 92 106 95 204 90 285 88

Example 9 Storage Stability ofGlutaraldehyde/Polyethylenimine-Crosslinked, Immobilized Acidovoraxfacilis 72 W Nitrilase Activity

[0075]Acidovorax facilis 72W cells (5% dry cell weight) were immobilizedin Pronova ISAGEL RG300 carrageenan (3.0 wt %) as described in Example7, and the resulting catalyst beads suspended in either aqueous 0.3 Mammonium bicarbonate or potassium bicarbonate at pH 7.3. The resultingimmobilized catalyst suspensions were stored at 5° C. in a capped glassbottle for 144 days, then samples of the catalyst beads assayed fornitrilase activity. The catalyst beads stored in 0.3 M ammoniumbicarbonate and 0.3 M potassium bicarbonate had 93% and 97% of theirinitial activities, respectively.

Example 10 Immobilization of E. coli SS 1001 in Carrageenan Beads WithGlutaraldehyde/Polyethylenimine-Crosslinking

[0076] Into a 250 mL media bottle equipped with magnetic stir bar andcontaining 64 g of water at 50° C. was slowly added 3.38 g ofcarrageenan with rapid stirring. The mixture was heated to 75-80° C.with rapid stirring until the carrageenan was completely dissolved, andthe resulting solution cooled to 55-56° C. (gelling temperature ca. 52°C.) in a thermostated water bath. A suspension of 24.5 g of E. coliSS1001 cells (wet cell weight) in 20.5 g of 0.30 M sodium phosphatebuffer (pH 7.3) was heated to 50° C. for 12 minutes, then added to thecarrageenan solution at 55-56° C. with stirring. The cell/carrageenanmixture was immediately added slowly to 450 mL of soybean oil at 50° C.with stirring using an overhead stirrer. After cell/carrageenan dropletsof the desired size were produced in the oil by controlling the stirringrate, the temperature of the oil was reduced to 35° C. to gel thedroplets, and the oil decanted from the resulting beads. The beads werewashed with 0.1 M potassium bicarbonate buffer (pH 7.0), then a 35 gportion of the beads was resuspended in 83 mL of this same buffer, and0.875 g of 25 wt % glutaraldehyde in water was added and the beads mixedfor 0.5 h at 25° C. To the mixture was then added 3.5 g of 12.5 wt %polyethylenimine (BASF Lupasol PR971L, average Mw ca. 750,000) in water,and the beads mixed for an additional hour at 25° C. The beads were thenwashed with 0.3 M ammonium bicarbonate (pH 7.3) and stored in this samebuffer at 5° C.

Example 11 Storage Stability ofGlutaraldehyde/Polyethylenimine-Crosslinked, Immobilized E. coli SS1001Nitrilase Activity

[0077]E. coli SS1001 cells (5% dry cell weight) were immobilized inPronova ISAGEL RG300 carrageenan (3.0 wt %) as in Example 10, and theresulting catalyst beads stored in 0.3 M ammonium bicarbonate at 5° C.in a capped glass bottle for 70 days. Assayed for nitrilase activityafter this storage period, a sample of the catalyst beads had 90% of theinitial nitrilase activity.

Example 12 Stability of Unimmobilized Acidovorax facilis Cell NitrilaseActivity in Ammonium Carbamate Storage Buffer

[0078]Acidovorax facilis 72W cells were isolated from cultured broth bycentrifugation, suspended at a concentration of 5% dry cell weight inaqueous ammonium carbamate (0.10 M or 0.30 M) at pH 7.3, and theresulting suspensions heated at 50° C. for 45 min to inactivate nitrilehydratase and amidase activities and stored at 5° C. Samples wereremoved from the cell suspensions over time and the samples assayed fornitrilase activity. Table 6 shows the relative nitrilase activity of thestored cell suspensions, with the nitrilase activity of the suspensionsat day 0 defined as 100%. Cells stored for 54 days in 0.10 M or 0.30 Mammonium carbamate retained a higher percentage of the initial nitrilaseactivity when compared to storage in either 0.10 M or 1.0 M sodiumacetate or potassium phosphate (Example 1). TABLE 6 nitrilase activity(%) (NH₄)HCO₂NH₂ days in storage 0.10 M 0.30 M 0 100 100 14 108 104 29109 95 43 101 91 54 101 90

Example 13 Immobilization of Acidovorax facilis 72W Cells in CalciumAlginate

[0079] Example 13 illustrates the immobilization of Acidovorax facilis72W (ATCC 55746) cells in both uncrosslinked and GA/PEI-crosslinkedcalcium alginate.

[0080] Into a 500-mL media bottle equipped with magnetic stir bar andcontaining 137.4 g of distilled, deionized water at 25° C. was slowlyadded 6.60 g of FMC BioPolymer Protanal® LF 10/60 alginate with rapidstirring. The mixture was heated to 75-80° C. with rapid stirring untilthe alginate was completely dissolved, and the resulting solution cooledto 25° C. A suspension of 78.3 g of Acidovorax facilis 72W wet cellpaste (23% dry cell weight) in 4.8 mL of 1.5 M sodium acetate buffer (pH7.0) and 12.9 mL of deionized water was added to the alginate solutionat 25° C. with stirring. The cell/alginate mixture was added dropwise bysyringe to 1280 mL of 0.10 M calcium acetate buffer (pH 7.0) at 25° C.with stirring. After stirring for 2 h, the buffer was decanted from theresulting beads, which were weighed (203 g of beads) and thenresuspended in 2.44 mL of decanted buffer per gram of beads. To prepareGA/PEI-crosslinked beads, 10.15 g of 25 wt % glutaraldehyde (GA) inwater was added with stirring and the beads mixed for 1.0 h at 25° C. Tothe suspension was then added 40.6 g of 12.5 wt % polyethylenimine (PEI)(BASF Lupasol® G35, average molecular weight ca. 20,000) in water, andthe beads mixed for an additional 20 h at 25° C. The crosslinked beadswere then washed twice with 1.0 L of 0.004 M calcium acetate buffer (pH7.0) at 25° C., and then stored in the desired storage buffer at 5° C.

[0081] PEI/GA-crosslinked beads were prepared by reversing the order ofaddition of GA and PEI. Uncrosslinked beads were prepared by theabove-described process except that GA and PEI were not added to thesuspension of beads in 0.10 M calcium acetate buffer.

Example 4 Stability of Immobilized Acidovorax facilis Nitrilase Activityin Ammonium Carbamate Storage Buffer

[0082]Acidovorax facilis 72W cells (7.5% dry cell weight) wereimmobilized in FMC BioPolymer Protanal® LF 10/60 alginate (2.75 wt %) asin Example 13 above, crosslinked with GA/PEI, PEI/GA, or notcrosslinked, and the resulting catalyst beads suspended in aqueous 0.10M or 0.30 M ammonium carbamate containing 4 mM calcium acetate at pH7.3. The resulting immobilized catalyst suspensions were stored at 5° C.in capped glass bottles, and samples were removed over time and theimmobilized Acidovorax facilis 72W cells assayed for nitrilase activity.

[0083] Table 7 shows the relative nitrilase activity of the storedimmobilized cell suspensions, with the activity at day 1 defined as100%. Immobilized cells stored in 0.10 M or 0.30 M ammonium carbamateretained a significantly-higher percentage of the initial nitrilaseactivity when compared to storage in 1.0 M potassium phosphate, ammoniumacetate, ammonium propionate, or in 50 mM potassium phosphate containing0.1 M ammonium chloride, at pH 7.3 and 5° C. Immobilized-cell catalystsstored for up to 68 days in 0.10 M or 0.30 M ammonium carbamate retaineda higher percentage of the initial nitrilase activity when compared tostorage in 0.1 M potassium phosphate, ammonium acetate, ammoniumpropionate, ammonium sulfate, or in 50 mM potassium phosphate containing1.0 M ammonium chloride, at pH 7.3 and 5° C. (Example 4). Table 8 liststhe change in optical density (OD) of the storage ammonium carbamatestorage buffers over this same time, and no significant microbialcontamination was observed. TABLE 7 nitrilase activity (%) (NH₄)HCO₂NH₂0.10 M, 0.30 M, PEI/GA GA/PEI 0.10 M, days in storage crosslinkedcrosslinked un-crosslinked 1 100 100 100 13 102 87 — 27 107 81 107 41113 79 122 54 117 — — 68 — 84 —

[0084] TABLE 8 OD (600 nm) (NH₄)HCO₂NH₂ 0.10 M, 0.30 M, days in PEI/GAGA/PEI 0.10 M, storage crosslinked crosslinked un-crosslinked 4 0.001 —— 6 — 0.005 0.007 21 0.006 — 0.042 27 — 0.009 — 33 — 0.010 — 38 — —0.110 40 0.025 — — 48 — 0.020 — 57 0.060 — — 65 — 0.006 —

What is claimed is:
 1. A method for preserving immobilized orunimmobilized microbial cells having nitrilase activity and forstabilizing the nitrilase activity thereof, the method comprising:adding to an aqueous suspension of immobilized or unimmobilizedmicrobial cells having nitrilase activity at least one compound selectedfrom the group consisting of inorganic carbonate salts and inorganicbicarbonate salts wherein the resulting total concentration of theinorganic salts in the aqueous suspension ranges from about 100 mM tothe saturation concentration of the inorganic salts.
 2. A method forpreserving immobilized or unimmobilized microbial cells having nitrilaseactivity and for stabilizing the nitrilase activity thereof, the methodcomprising: adding to an aqueous suspension of immobilized orunimmobilized microbial cells having nitrilase activity at least onecompound selected from the group consisting of inorganic carbamatesalts, wherein the resulting total concentration of the inorganic saltsin the aqueous suspension ranges from about 100 mM to the saturationconcentration of the inorganic salts.
 3. The method of claim 2 whereinthe microbial cells having nitrilase activity are selected from thegroup consisting of Acidovorax facilis 72W (ATCC 55746), Acidovoraxfacilis 72-PF-15 (ATCC 55747), Acidovorax facilis 72-PF-17 (ATCC 55745),and microbial cells transformed with Acidovorax facilis 72W nitrilaseactivity.
 4. The method of claim 3 wherein the microbial cellstransformed with Acidovorax facilis 72W nitrilase activity are selectedfrom the group consisting of E. coli SS1001 (ATCC PTA-1177) and E. coliSW91 (ATCC PTA-1175).
 5. The method of claim 2, wherein the inorganiccarbamate salt is ammonium carbamate.
 6. The method of claim 2, whereinthe pH of the aqueous suspension of immobilized or unimmobilizedmicrobial cells having nitrilase activity ranges from about pH 6 toabout pH
 10. 7. The method of claim 2 wherein the temperature of theaqueous suspension of immobilized or unimmobilized microbial cellsranges from about 0° C. to about 45° C.
 8. The method of claim 5 whereinthe microbial cells are immobilized in polyacrylamide gel, alginate, orcarrageenan.
 9. The method of claim 8 wherein the microbial cells arefirst immobilized in carrageenan or alginate, then crosslinked withglutaraldehyde and polyethylenimine.
 10. The method of claim 8 whereinthe resulting total concentration of ammonium carbamate in the aqueoussuspension ranges from about 100 mM to about 1000 mM.
 11. The method ofclaim 8 wherein the microbial cells are immobilized in alginate, and theresulting total concentration of inorganic carbamate salt in the aqueoussuspension ranges from about 100 mM to about 1000 mM.
 12. The method ofclaim 8 further comprising adding to the aqueous suspension a calciumsalt of at least 2 mM, and wherein the microbial cells are immobilizedin alginate.
 13. A method for preserving immobilized microbial cellshaving nitrilase activity and for stabilizing the nitrilase activitythereof, the method comprising: adding ammonium carbamate and at least 2mM calcium acetate or calcium chloride to an aqueous suspension ofalginate-immobilized microbial cells having nitrilase activity andselected from the group consisting of Acidovorax facilis 72W (ATCC55746), Acidovorax facilis 72-PF-15 (ATCC 55747), Acidovorax facilis72-PF-17 (ATCC 55745), and microbial cells transformed with Acidovoraxfacilis 72W nitrilase activity, wherein the resulting aqueous suspensionhas a) a total concentration of the ammonium carbamate ranging fromabout 100 mM to 1000 mM, and b) a pH from about pH 6 to about pH
 10. 14.The method of claim 13 wherein the microbial cells transformed withAcidovorax facilis 72W nitrilase activity are E. coli SS1001 (ATCCPTA-1177) or E. coli SW91 (ATCC PTA-1175).
 15. The method of claim 13wherein the microbial cells are immobilized in alginate, thencrosslinked with glutaraldehyde and polyethylenimine.